PROJECT 2 PROJECT SUMMARY/ABSTRACT Herpesvirus miRNAs target many different cellular and viral processes involved in viral immune recognition, apoptosis, cell cycle regulation, cellular trafficking as well as viral latency and lytic replication. We have made significant advances elucidating functional roles for HCMV miRNAs including the identification of HCMV miRNAs that regulate HCMV IE1 as well as multiple cellular genes involved in cell cycle modulation, formation of the viral assembly complex, and TLR2 signaling. Importantly, we have observed that the HCMV miRNAs work coordinately to efficiently down-regulate individual genes and that the HCMV miRNAs target multiple individual genes within the same cellular pathways. Recently, we have determined that several HCMV miRNAs target multiple members of the Epidermal Growth Factor Receptor (EGFR) signaling pathways that are important in viral latency and hematopoiesis. We have observed that a double HCMV mutation of two of the miRNAs targeting this pathway results in an increase in viral reactivation while two other viral miRNA double mutants fail to reactivate in a human progenitor cell (HPC) culture system in vitro. Lastly, EGFR signaling has also been shown to be important in HPC hematopoiesis. We have observed that infection of HPCs with single and double HCMV miRNA mutations results in reduced levels of CFU-GM, CFU-GEMM and BFU-E colony formation as well as myelosuppression in comparison to WT HCMV, suggesting a role for the miRNAs in hematopoiesis. Therefore, we hypothesize that HCMV miRNA regulation of EGFR signaling plays a critical role in viral latency and reactivation as well as hematopoiesis. In the current proposal we will characterize the EGFR HCMV miRNA targetome and the functional relevance of the EGFR miRNA targets for viral latency and replication in CD34+ HPC in vitro. These studies will be extended in a newly developed human fetal bone, liver and thymus (BLT) NOD-scidIL2Rgc null mouse model that is able to support latent HCMV infection as well as reactivation from latency (Core A). We will determine how the HCMV miRNAs that target this pathway interphase with UL133/8 EGFR regulation in the establishment and maintenance of viral latency in Project 1. Additionally, several of the HCMV miRNAs also target genes activated by the HCMV latency gene US28 and UL7. We will examine the role that these miRNAs play in regulating US28 signaling and US28-EGFR signaling cross-talk in Project 3 and UL7 (Project 4). Lastly, we will determine the contribution of the HCMV EGFR miRNAs in hematopoiesis in comparison to results of WT HCMV in Project 5.